The attachment of cells to extracellular matrix (ECM) is crucial for a variety of physiological and pathological processes. This interaction (cell adhesion) is mediated primarily by integrins, a group of heterodimeric transmembrane receptors that bind to ECM proteins via their extracellular domains. Upon ECM engagement, integrins cluster and transduce signals into intracellular compartment leading to the formation of large protein complexes called focal adhesions (FAs) that connect integrin cytoplasmic tails (CTs) to the actin cytoskeleton. This latter step, i.e., the formation of FAs and their linkage to actin, promotes firm cell adhesion. Furthermore, it allows regulation of dynamic adhesive processes such as cell spreading and migration. Our long term goal is to obtain a detailed molecular understanding of FAs and to elucidate how they are connected to actin and modulated during various adhesive processes. To this end, we have been focusing on a major component of FAs - integrin-linked kinase (ILK). Originally discovered as an integrin linking protein that binds to integrin 2 CTs, ILK has been established as a multifunctional protein that transmits diverse mechanical and biochemical signals between integrins and actin. A key initial step for ILK function is its tight binding to PINCH - a LIM- containing adaptor. This interaction not only promotes the localization of ILK to integrin adhesion sites but also creates a stable platform that harbors many proteins to regulate dynamic FA assembly and diverse signaling pathways. Over the past several years, we have made a major progress towards building a molecular landscape of the ILK/PINCH network and showed how it functions in a spatiotemporal manner in various cellular processes. In collaboration with clinical scientists, we have also shown that the ILK/PINCH complex is abnormally elevated in failing human hearts, suggesting its direct involvement in cardiac dysfunction. Coincidently, a recent study in mice has shown that a G-actin sequestering peptide, thymosin beta-4 (tb4), may repair cardiac damage by modulating the ILK/PINCH-mediated cell migration and survival. While this has led to widespread follow-up investigations and the launching of a tb4-based phase1A clinical trial on treating heart injury patients, the underlying molecular mechanism remains obscure. In preliminary investigation, we have discovered a novel ILK/PINCH-mediated integrin-actin linkage that may be crucial for cell migration and survival. This linkage appears to be dynamically regulated by tb4. In the next phase of our study, we will use multidisciplinary structural/functional approach to vigorously investigate this linkage and its regulation by tb4. The studies will lead to a new paradigm for understanding the ILK/PINCH-mediated cell adhesion. They will also impact on the tb4-based therapy of cardiac disorder and possibly other diseases. PUBLIC HEALTH RELEVANCE: The heterocomplex between integrin-linked kinase (ILK) and LIM-only adaptor PINCH plays a central role in transmitting information between extracellular matrix and actin cytoskeleton. Dysregulation of this complex has been recently linked to heart attack and its regulation by a naturally occurring human peptide, thymosin beta-4, has been shown to exert therapeutic effect in mouse models. Our proposal will elucidate the molecular basis of the ILK/PINCH-mediated ECM/actin linkage and how it is regulated by tb4, which may lead to fundamental understanding of the ILK/PINCH function and also impact on tb4-based therapy of heart disease.